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Bacteriophage T7 DNA primase recognizes 5′-GTC-3′ in single-stranded DNA. The primase contains a single Cys 4 zinc-binding motif that is essential for recognition. Biochemical and mutagenic analyses suggest that the Cys 4 motif contacts cytosine of 5′-GTC-3′ and may also contribute to thymine recognition. Residues His 33 and Asp 31 are critical for these interactions. Biochemical analysis also reveals that T7 primase selectively binds CTP in the absence of DNA. We propose that bound CTP selects the remaining base G, of 5′-GTC-3′, by base pairing. Our deduced mechanism for recognition of ssDNA by Cys 4 motifs bears little resemblance to the recognition of trinucleotides of double-stranded DNA by Cys 2 His 2 zinc fingers.

Two colinear bacteriophage T7 gene 4 proteins provide helicase and primase functions in vivo. T7 primase differs from T7 helicase by an additional 63 residues at the amino terminus. This terminal domain contains a zinc-binding motif which mediates an interaction with the basic primase recognition sequence 3'-CTG-5'. We have generated a chimeric primase in which the 81 amino-terminal residues are derived from the primase of phage T3 and the 484 carboxyl-terminal residues are those of phage T7 helicase. The amino-terminal domain of T3 primase is 50% homologous with that of T7 primase. The resulting T3/T7 chimeric protein is a functional primase in vivo. While the primase activity of the purified protein is about one-third that of T7 primase, the recognition sites used and the oligoribonucleotides synthesized from these sites are identical. We conclude that the residues responsible for the interaction with the sequence 3'-CTG-5' are conserved between the chimeric and T7 proteins.